Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids

ABSTRACT

The present invention provides a method of PCR, methylation specific PCR (MSP), for rapid identification of DNA methylation patterns in a CpG-containing nucleic acid. MSP uses agents to modify unmethylated cytosine in a nucleic acid of interest, and then uses the PCR reaction to amplify the CpG-containing nucleic acid in the specimen by means of CpG-specific oligonucleotide primers. The oligonecleotide primers distinguish between modified methylated and nonmethylated nucleic acid. Kits utilizing MSP for the detection of methylated CpG-containing nucleic acids are also provided.

This invention was made with government support under Grant Nos. CA43318and CA54396 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to regulation of geneexpression, and more specifically to a method of determining the DNAmethylation status of CpG sites in a given locus.

BACKGROUND OF THE INVENTION

In higher order eukaryotes DNA is methylated only at cytosines located5' to guanosine in the CpG dinucleotide. This modification has importantregulatory effects on gene expression, especially when involving CpGrich areas, known as CpG islands, located in the promoter regions ofmany genes. While almost all gene-associated islands are protected frommethylation on autosomal chromosomes, extensive methylation of CpGislands has been associated with transcriptional inactivation ofselected imprinted genes and genes on the inactive X -chromosome offemales. Abberant methylation of normally unmethylated CpG islands hasbeen described as a frequent event in immortalized and transformedcells, and has been associated with transcriptional inactivation ofdefined tumor suppressor genes in human cancers.

Human cancer cells typically contain somatically altered genomes,characterized by mutation, amplification, or deletion of critical genes.In addition, the DNA template from human cancer cells often displayssomatic changes in DNA methylation (E. R. Fearon, et al., Cell, 61:759,1990; P. A. Jones, et al., Cancer Res., 46:461, 1986; R. Holliday,Science, 238:163, 1987; A. De Bustros, et aL, Proc. Natl. Acad. Sci.,USA, 85:5693, 1988); P. A. Jones, et al., Adv. Cancer Res., 54:1, 1990;S. B. Baylin, et al., Cancer Cells, 3 :383, 1991; M. Makos, et al, Proc.Natl. Acad. Sci., USA, 89:1929, 1992; N. Ohtani-Fujita, et al.,Oncogene, 8:1063, 1993). However, the precise role of abnormal DNAmethylation in human tumorigenesis has not been established. DNAmethylases transfer methyl groups from the universal methyl donorS-adenosyl methionine to specific sites on the DNA. Several biologicalfunctions have been attributed to the methylated bases in DNA. The mostestablished biological function is the protection of the DNA fromdigestion by cognate restriction enzymes. The restriction modificationphenomenon has, so far, been observed only in bacteria. Mammalian cells,however, possess a different methylase that exclusively methylatescytosine residues on the DNA, that are 5' neighbors of guanine (CpG).This methylation has been shown by several lines of evidence to play arole in gene activity, cell differentiation, tumorigenesis, X-chromosomeinactivation, genomic imprinting and other major biological processes(Razin, A., H., and Riggs, R. D. eds. in DNA Methylation Biochemistryand Biological Significance, Springer-Verlag, N.Y., 1984).

A CpG rich region, or "CpG island", has recently been identified at17p13.3, which is aberrantly hypermethylated in multiple common types ofhuman cancers (Makos, M., et al., Proc. Natl. Acad. Sci. USA, 89:1929,1992; Makos, M., et al, Cancer Res., 53:2715, 1993; Makos, M., et al,Cancer Res. 53:2719, 1993). This hypermethylation coincides with timingand frequency of 17p losses and p53 mutations in brain, colon, and renalcancers. Silenced gene transcription associated with hypermethylation ofthe normally unmethylated promoter region CpG islands has beenimplicated as an alternative mechanism to mutations of coding regionsfor inactivation of tumor suppressor genes (Baylin, S. B., et al, CancerCells, 3:383, 1991; Jones, P. A. and Buckley, J. D., Adv. Cancer Res.,54:1-23, 1990). This change has now been associated with the loss ofexpression of VHL, a renal cancer tumor suppressor gene on 3p (J. G.Herman, et al, Proc. Natl. Acad. Sci. USA, 91:9700-9704, 1994), theestrogen receptor gene on 6q (Ottaviano, Y. L., et al., Cancer Res.,54:2552, 1994) and the H19 gene on 11p (Steenman, M. J. C., et al,Nature Genetics, 7:433, 1994).

In eukaryotic cells, methylation of cytosine residues that areimmediately 5' to a guanosine, occurs predominantly in CG poor regions(Bird, A., Nature, 321:209, 1986). In contrast, discrete regions of CGdinucleotides called CpG islands remain unmethylated in normal cells,except during X-chromosome inactivation (Migeon, et al., supra) andparental specific imprinting (Li, et al, Nature, 366:362, 1993) wheremethylation of 5' regulatory regions can lead to transcriptionalrepression. De novo methylation of the Rb gene has been demonstrated ina small fraction of retinoblastomas (Sakai, et al, Am. J. Hum. Genet.,48:880, 1991), and recently, a more detailed analysis of the VHL geneshowed aberrant methylation in a subset of sporadic renal cellcarcinomas (Herman, et al., Proc. Natl. Acad. Sci., U.S.A., 91:9700,1994). Expression of a tumor suppressor gene can also be abolished by denovo DNA methylation of a normally unmethylated 5' CpG island (Issa, etal, Nature Genet., 7:536, 1994; Herman, et al., supra; Merlo, et al.,Nature Med., 1:686, 1995; Herman, et al., Cancer Res., 56:722, 1996;Graff, et al, Cancer Res., 55:5195, 1995; Herman, et al, Cancer Res.,55:4525, 1995).

Most of the methods developed to date for detection of methylatedcytosine depend upon cleavage of the phosphodiester bond alongsidecytosine residues, using either methylation-sensitive restrictionenzymes or reactive chemicals such as hydrazine which differentiatebetween cytosine and its 5-methyl derivative. The use ofmethylation-sensitive enzymes suffers from the disadvantage that it isnot of general applicability, since only a limited proportion ofpotentially methylated sites in the genome can be analyzed. Genomicsequencing protocols which identify a 5-MeC residue in genomic DNA as asite that is not cleaved by any of the Maxam Gilbert sequencingreactions, are a substantial improvement on the original genomicsequencing method, but still suffer disadvantages such as therequirement for large amount of genomic DNA and the difficulty indetecting a gap in a sequencing ladder which may contain bands ofvarying intensity.

Mapping of methylated regions in DNA has relied primarily on Southernhybridization approaches, based on the inability ofmethylation-sensitive restriction enzymes to cleave sequences whichcontain one or more methylated CpG sites. This method provides anassessment of the overall methylation status of CpG islands, includingsome quantitative analysis, but is relatively insensitive, requireslarge amounts of high molecular weight DNA and can only provideinformation about those CpG sites found within sequences recognized bymethylation-sensitive restriction enzymes. A more sensitive method ofdetecting methylation patterns combines the use of methylation-sensitiveenzymes and the polymerase chain reaction (PCR). After digestion of DNAwith the enzyme, PCR will amplify from primers flanking the restrictionsite only if DNA cleavage was prevented by methylation. LikeSouthern-based approaches, this method can only monitor CpG methylationin methylation-sensitive restriction sites. Moreover, the restriction ofunmethylated DNA must be complete, since any uncleaved DNA will beamplified by PCR yielding a false positive result for methylation. Thisapproach has been useful in studying samples where a high percentage ofalleles of interest are methylated, such as the study of imprinted genesand X-chromosome inactivated genes. However, difficulties indistinguishing between incomplete restriction and low numbers ofmethylated alleles make this approach unreliable for detection of tumorsuppressor gene hypermethylation in small samples where methylatedalleles represent a small fraction of the population.

Another method that avoids the use of restriction endonucleases utilizesbisulfite treatment of DNA to convert all unmethylated cytosines touracil. The altered DNA is amplified and sequenced to show themethylation status of all CpG sites. However, this method is technicallydifficult, labor intensive and without cloning amplified products, it isless sensitive than Southern analysis, requiring approximately 10% ofthe alleles to be methylated for detection.

Identification of the earliest genetic changes in tumorigenesis is amajor focus in molecular cancer research. Diagnostic approaches based onidentification of these changes are likely to allow implementation ofearly detection strategies and novel therapeutic approaches targetingthese early changes might lead to more effective cancer treatment.

SUMMARY OF THE INVENTION

The precise mapping of DNA methylation patterns in CpG islands hasbecome essential for understanding diverse biological processes such asthe regulation of imprinted genes, X-chromosome inactivation, and tumorsuppressor gene silencing in human cancer. The present inventionprovides a method for rapid assessment of the methylation status of anygroup of CpG sites within a CpG island, independent of the use ofmethylation-sensitive restriction enzymes.

The method of the invention includes modification of DNA by sodiumbisulfite or a comparable agent which converts all unmethylated but notmethylated cytosines to uracil, and subsequent amplification withprimers specific for methylated versus unmethylated DNA. This method of"methylation specific PCR" or MSP, requires only small amounts of DNA,is sensitive to 0.1% of methylated alleles of a given CpG island locus,and can be performed on DNA extracted from paraffin-embedded samples,for example. MSP eliminates the false positive results inherent toprevious PCR-based approaches which relied on differential restrictionenzyme cleavage to distinguish methylated from unmethylated DNA.

In a particular aspect of the invention, MSP is useful for identifyingpromoter region hypermethylation changes associated with transcriptionalinactivation in tumor suppressor genes, for example, p16, p15,E-cadherin and VHL, in human neoplasia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows genomic sequencing of p16. The sequence shown has the most5' region at the bottom of the gel, beginning at +175 in relation to amajor transcriptional start site (Hara, et al., Mol. Cell Biol, 16:859,1996). All cytosines in the unmethylated cell line H249 have beenconverted to thymidine, while all C's in CpG dinucleotides in themethylated cell H157 remains as C, indicating methylation. ! enclosed aBstUI site which is at -59 in relation to the transnational start sitein Genbank sequence U12818 (Hussussian, et al, Nat. Genet., 8:15, 1994),but which is incorrectly identified as CGCA in sequence X94154 (Hara, etal., supra). This CGCG site represents the 3' location of the senseprimer used for p16 MSP.

FIGS. 2A-2E show polyacrylamide gels with the Methylation Specific PCRproducts of p16. Primer sets used for amplification are designated asunmethylated (U), methylated (M), or unmodified/wild-type (W). *designates the molecular weight marker pBR322-MspI digest. Panel A showsamplification of bisulfite-treated DNA from cancer cell lines and normallymphocytes, and untreated DNA (from cell line H249). Panel B showsmixing of various amount of H157 DNA with 1 μg of H249 DNA prior tobisulfite treatment to assess the detection sensitivity of MSP formethylated alleles. Modified DNA from a primary lung cancer sample andnormal lung are also shown. Panel C shows amplification with the p16-U2(U) primers, and p16-M2 (M) described in Table 1. Panel D shows theamplified p16 products of panel C restricted with BstUI(+) or notrestricted (-). Panel E shows results of testing for regionalmethylation of CpG islands with MSP, using sense primers p16-U2 (U) andp16-M2 (M), which are methylation specific, and an antisense primerwhich is not methylation specific.

FIGS. 3A-3E show polyacrylaride gels of MSP products from analysis ofseveral genes. Primer sets used for amplification are not designated asunmethylated (U), methylatecd (M), or unmodified/wild-type (W). *designates the molecular weight marker pBR322-MspI digest and **designates the 123 bp molecular weight marker. All DNA samples werebisulfite treated except those designated untreated. Panel A shows theresults from MSP for p15. Panel B shows the p15 products restricted withBstUI (+) or not restricted (-). Panel C shows the products of MSP forVHL. Panel D shows the VHL products restricted with BstUI(+) or notrestricted (-). Panel E shows the products of MSP for E-cadherin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methylation specific PCR (MSP) foridentification of DNA methylation patterns. MSP uses the PCR reactionitself to distinguish between methylated and unmethylated DNA, whichadds an improved sensitivity of methylation detection.

Unlike previous genomic sequencing methods for methylationidentification which utilizes amplification primers which arespecifically designed to avoid the CpG sequences, MSP primers arespecifically designed to recognize CpG sites to take advantage of thedifferences in methylation to amplify specific products to be identifiedby the invention assay.

As illustrated in the Examples below, MSP provides significantadvantages over previous PCR and other methods used for assayingmethylation. MSP is markedly more sensitive than Southern analyses,facilitating detection of low numbers of methylated alleles and thestudy of DNA from small samples. MSP allows the study ofparaffin-embedded materials, which could not previously be analyzed bySouthern analysis. MSP also allows examination of all CpG sites, notjust those within sequences recognized by methylation-sensitiverestriction enzymes. This markedly increases the number of such siteswhich can be assessed and will allow rapid, fine mapping of methylationpatterns throughout CpG rich regions. MSP also eliminates the frequentfalse positive results due to partial digestion of methylation-sensitiveenzymes inherent in previous PCR methods for detecting methylation.Furthermore, with MSP, simultaneous detection of unmethylated andmethylated products in a single sample confirms the integrity of DNA asa template for PCR and allows a semi-quantitative assessment of alleletypes which correlates with results of Southern analysis. Finally, theability to validate the amplified product by differential restrictionpatterns is an additional advantage.

The only technique that can provide more direct analysis than MSP formost CpG sites within a defined region is genomic sequencing. However,MSP can provide similar information and has the following advantages.First, MSP is much simpler and requires less than genomic sequencing,with a typical PCR and gel analysis taking 4-6 hours. In contrast,genomic sequencing, amplification, cloning, and subsequent sequencingmay take days. MSP also avoids the use of expensive sequencing reagentsand the use of radioactivity. Both of these factors make MSP bettersuited for the analysis of large numbers of samples. Third, the use ofPCR as the step to distinguish methylated from unmethylated DNA in MSPallows for significant increase in the sensitivity of methylationdetection. For example, if cloning is not used prior to genomicsequencing of the DNA, less than 10% methylated DNA in a background ofunmethylated DNA cannot be seen (Myohanen, et al, supra). The use of PCRand cloning does allow sensitive detection of methylation patterns invery small amounts of DNA by genomic sequencing (Frommer, et al, Proc.Natl Acad. Sci. USA, 89:1827, 1992; Clark, et al, Nucleic AcidsResearch, 22:2990, 1994). However, this means in practice that it wouldrequire sequencing analysis of 10 clones to detect 10% methylation, 100clones to detect 1% methylation, and to reach the level of sensitivitywe have demonstrated with MSP (1:1000), one would have to sequence 1000individual clones.

In a first embodiment, the invention provides a method for detecting amethylated CpG-containing nucleic acid, the method including contactinga nucleic acid-containing specimen with an agent that modifiesunmethylated cytosine; amplifying the CpG-containing nucleic acid in thespecimen by means of CpG-specific oligonucleotide primers; and detectingthe methylated nucleic acid. It is understood that while theamplification step is optional, it is desirable in the preferred methodof the invention.

The term "modifies" as used herein means the conversion of anunmethylated cytosine to another nucleotide which will distinguish theunmethylated from the methylated cytosine. Preferably, the agentmodifies unmethylated cytosine to uracil. Preferably, the agent used formodifying unmethylated cytosine is sodium bisulfite, however, otheragents that similarly modify unmethylated cytosine, but not methylatedcytosine can also be used in the method of the invention. Sodiumbisulfite (NaHSO₃) reacts readily with the 5,6-double bond of cytosine,but poorly with methylated cytosine. Cytosine reacts with the bisulfiteion to form a sulfonated cytosine reaction intermediate which issusceptible to deamination, giving rise to a sulfonated uracil. Thesulfonate group can be removed under alkaline conditions, resulting inthe formation of uracil. Uracil is recognized as a thymine by Taqpolymerase and therefore upon PCR, the resultant product containscytosine only at the position where 5-methylcytosine occurs in thestarting template DNA.

The primers used in the invention for amplification of theCpG-containing nucleic acid in the specimen, after bisulfitemodification, specifically distinguish between untreated DNA,methylated, and non-methylated DNA. MSP primers for the non-methylatedDNA preferably have a T in the 3' CG pair to distinguish it from the Cretained in methylated DNA, and the compliment is designed for theantisense primer. MSP primers usually contain relatively few Cs or Gs inthe sequence since the Cs will be absent in the sense primer and the Gsabsent in the antisense primer (C becomes modified to U (uracil) whichis amplified as T (thymidine) in the amplification product).

The primers of the invention embrace oligonucleotides of sufficientlength and appropriate sequence so as to provide specific initiation ofpolymerization on a significant number of nucleic acids in thepolymorphic locus. Specifically, the term "primer" as used herein refersto a sequence comprising two or more deoxyribonucleotides orribonucleotides, preferably more than three, and most preferably morethan 8, which sequence is capable of initiating synthesis of a primerextension product, which is substantially complementary to a polymorphiclocus strand. Environmental conditions conducive to synthesis includethe presence of nucleoside triphosphates and an agent forpolymerization, such as DNA polymerase, and a suitable temperature andpH. The primer is preferably single stranded for maximum efficiency inamplification, but may be double stranded. If double stranded, theprimer is first treated to separate its strands before being used toprepare extension products. Preferably, the primer is an oligodeoxyribonucleo-tide. The primer must be sufficiently long to prime thesynthesis of extension products in the presence of the inducing agentfor polymerization. The exact length of primer will depend on manyfactors, including temperature, buffer, and nucleotide composition. Theoligonucleotide primer typically contains 12-20 or more nucleotides,although it may contain fewer nucleotides.

Primers of the invention are designed to be "substantially"complementary to each strand of the genomic locus to be amplified andinclude the appropriate G or C nucleotides as discussed above. Thismeans that the primers must be sufficiently complementary to hybridizewith their respective strands under conditions which allow the agent forpolymerization to perform. In other words, the primers should havesufficient complementarity with the 5' and 3' flanking sequences tohybridize therewith and permit amplification of the genomic locus.

Oligonucleotide primers of the invention are employed in theamplification process which is an enzymatic chain reaction that producesexponential quantities of target locus relative to the number ofreaction steps involved. Typically, one primer is complementary to thenegative (-) strand of the locus and the other is complementary to thepositive (+) strand. Annealing the primers to denatured nucleic acidfollowed by extension with an enzyme, such as the large fragment of DNAPolymerase I (Klenow) and nucleotides, results in newly synthesized +and - strands containing the target locus sequence. Because these newlysynthesized sequences are also templates, repeated cycles of denaturing,primer annealing, and extension results in exponential production of theregion (i.e., the target locus sequence) defined by the primer. Theproduct of the chain reaction is a discrete nucleic acid duplex withtermini corresponding to the ends of the specific primers employed.

The oligonucleotide primers of the invention may be prepared using anysuitable method, such as conventional phosphotriester and phosphodiestermethods or automated embodiments thereof. In one such automatedembodiment, diethylphosphoramidites are used as starting materials andmay be synthesized as described by Beaucage, et al. (TetrahedronLetters, 22:1859-1862, 1981). One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066.

Any nucleic acid specimen, in purified or nonpurified form, can beutilized as the starting nucleic acid or acids, provided it contains, oris suspected of containing, the specific nucleic acid sequencecontaining the target locus (e.g., CpG). Thus, the process may employ,for example, DNA or RNA, including messenger RNA, wherein DNA or RNA maybe single stranded or double stranded. In the event that RNA is to beused as a template, enzymes, and/or conditions optimal for reversetranscribing the template to DNA would be utilized. In addition, aDNA-RNA hybrid which contains one strand of each may be utilized. Amixture of nucleic acids may also be employed, or the nucleic acidsproduced in a previous amplification reaction herein, using the same ordifferent primers may be so utilized. The specific nucleic acid sequenceto be amplified, i.e., the target locus, may be a fraction of a largermolecule or can be present initially as a discrete molecule, so that thespecific sequence constitutes the entire nucleic acid. It is notnecessary that the sequence to be amplified be present initially in apure form; it may be a minor fraction of a complex mixture, such ascontained in whole human DNA.

The nucleic acid-containing specimen used for detection of methylatedCpG may be from any source including brain, colon, urogenital,hematopoietic, thymus, testis, ovarian, uterine, prostate, breast,colon, lung and renal tissue and may be extracted by a variety oftechniques such as that described by Maniatis, et al (Molecular Cloning:A Laboratory Manual, Cold Spring Harbor, N.Y., pp 280, 281, 1982).

If the extracted sample is impure (such as plasma, serum, or blood or asample embedded in parrafin), it may be treated before amplificationwith an amount of a reagent effective to open the cells, fluids,tissues, or animal cell membranes of the sample, and to expose and/orseparate the strand(s) of the nucleic acid(s). This lysing and nucleicacid denaturing step to expose and separate the strands will allowamplification to occur much more readily.

Where the target nucleic acid sequence of the sample contains twostrands, it is necessary to separate the strands of the nucleic acidbefore it can be used as the template. Strand separation can be effectedeither as a separate step or simultaneously with the synthesis of theprimer extension products. This strand separation can be accomplishedusing various suitable denaturing conditions, including physical,chemical, or enzymatic means, the word "denaturing" includes all suchmeans. One physical method of separating nucleic acid strands involvesheating the nucleic acid until it is denatured. Typical heatdenaturation may involve temperatures ranging from about 80° to 105° C.for times ranging from about 1 to 10 minutes. Strand separation may alsobe induced by an enzyme from the class of enzymes known as helicases orby the enzyme RecA, which has helicase activity, and in the presence ofriboATP, is known to denature DNA. The reaction conditions suitable forstrand separation of nucleic acids with helicases are described by KuhnHoffmann-Berling (CSH-Quantitative Biology, 43:63, 1978) and techniquesfor using RecA are reviewed in C. Radding (Ann. Rev. Genetics,16:405-437, 1982).

When complementary strands of nucleic acid or acids are separated,regardless of whether the nucleic acid was originally double or singlestranded, the separated strands are ready to be used as a template forthe synthesis of additional nucleic acid strands. This synthesis isperformed under conditions allowing hybridization of primers totemplates to occur. Generally synthesis occurs in a buffered aqueoussolution, preferably at a pH of 7-9, most preferably about 8.Preferably, a molar excess (for genomic nucleic acid, usually about 10⁸:1 primer:template) of the two oligonucleotide primers is added to thebuffer containing the separated template strands. It is understood,however, that the amount of complementary strand may not be known if theprocess of the invention is used for diagnostic applications, so thatthe amount of primer relative to the amount of complementary strandcannot be determined with certainty. As a practical matter, however, theamount of primer added will generally be in molar excess over the amountof complementary strand (template) when the sequence to be amplified iscontained in a mixture of complicated long-chain nucleic acid strands. Alarge molar excess is preferred to improve the efficiency of theprocess.

The deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP areadded to the synthesis mixture, either separately or together with theprimers, in adequate amounts and the resulting solution is heated toabout 90°-100° C. from about 1 to 10 minutes, preferably from 1 to 4minutes. After this heating period, the solution is allowed to cool toroom temperature, which is preferable for the primer hybridization. Tothe cooled mixture is added an appropriate agent for effecting theprimer extension reaction (called herein "agent for polymerization"),and the reaction is allowed to occur under conditions known in the art.The agent for polymerization may also be added together with the otherreagents if it is heat stable. This synthesis (or amplification)reaction may occur at room temperature up to a temperature above whichthe agent for polymerization no longer functions. Thus, for example, ifDNA polymerase is used as the agent, the temperature is generally nogreater than about 40° C. Most conveniently the reaction occurs at roomtemperature.

The agent for polymerization may be any compound or system which willfunction to accomplish the synthesis of primer extension products,including enzymes. Suitable enzymes for this purpose include, forexample, E. coli DNA polymerase I, Klenow fragment of E. coli DNApolymerase I, T4 DNA polymerase, other available DNA polymerases,polymerase muteins, reverse transcriptase, and other enzymes, includingheat-stable enzymes (i.e., those enzymes which perform primer extensionafter being subjected to temperatures sufficiently elevated to causedenaturation). Suitable enzymes will facilitate combination of thenucleotides in the proper manner to form the primer extension productswhich are complementary to each locus nucleic acid strand. Generally,the synthesis will be initiated at the 3' end of each primer and proceedin the 5' direction along the template strand, until synthesisterminates, producing molecules of different lengths. There may beagents for polymerization, however, which initiate synthesis at the 5'end and proceed in the other direction, using the same process asdescribed above.

Preferably, the method of amplifying is by PCR, as described herein andas is commonly used by those of ordinary skill in the art. Alternativemethods of amplification have been described and can also be employed aslong as the methylated and non-methylated loci amplified by PCR usingthe primers of the invention is similarly amplified by the alternativemeans.

The amplified products are preferably identified as methylated ornon-methylated by sequencing. Sequences amplified by the methods of theinvention can be further evaluated, detected, cloned, sequenced, and thelike, either in solution or after binding to a solid support, by anymethod usually applied to the detection of a specific DNA sequence suchas PCR, oligomer restriction (Saiki, et al, BiolTechnology, 3:1008-1012,1985), allele-specific oligonucleotide (ASO) probe analysis (Conner, etal., Proc. Natl. Acad. Sci. USA, 80:278, 1983), oligonucleotide ligationassays (OLAs) (Landegren, et al., Science, 241:1077, 1988), and thelike. Molecular techniques for DNA analysis have been reviewed(Landegren, et al., Science, 242:229-237, 1988).

Optionally, the methylation pattern of the nucleic acid can be confirmedby restriction enzyme digestion and Southern blot analysis. Examples ofmethylation sensitive restriction endonucleases which can be used todetect 5'CpG methylation include SmaI, SacII, EagI, MspI, HpaII, BstUIand BssHII, for example.

Exemplaiy target polynucleotide sequences to which the primer hybridizeshave a sequence as listed below.

    __________________________________________________________________________                                       SEQ ID NO.                                 __________________________________________________________________________    Wild type p16                                                                             5'-GCGGTCCGCCCCACCCTCTG-3';                                                                          1                                                      5'-CCACGGCCGCGGCCCG-3';                                                                              2                                          Methylated p16-1*                                                                         5'-GCGATCCGCCCCACCCTCTAATAA-3';                                                                      3                                                      5'-TTACGGTCGCGGTTCGGGGTC-3';                                                                         4                                          Unmethylated p16-1                                                                        5'-ACAATCCACCCCACCCTCTAATAA-3';                                                                      5                                                      5'-TTATGGTTGTGGTTTGGGGTTG-3';                                                                        6                                          Methylated p16-2                                                                          5'-GCGATCCGCCCCACCCTCTAATAA-3'                                                                       7                                                      5'-CGGTCGGAGGTCGATTTAGGTGG-3'                                                                        8                                          Unmethylated p16-2                                                                        5'-ACAATCCACCCCACCCTCTAATAA-3';                                                                      9                                                      5'-TGGTTGGAGGTTGATTTAGGTGG-3';                                                                       10                                         Wild type p15                                                                             5'-TCTGGCCGCAGGGTGCG-3';                                                                             11                                                     5'-CCGGCCGCTCGGCCACT-3';                                                                             12                                         Methylated p15                                                                            5'-AACCGCAAAATACGAACGC-3';                                                                           13                                                     5'-TCGGTCGTTCGGTTATTGTACG-3';                                                                        14                                         Unmethylated p15                                                                          5'-AACCACAAAATACAAACACATCACA-3';                                                                     15                                                     5'-TTGGTTGTTTGGTTATTGTATGG-3';                                                                       16                                         Methylated VHL                                                                            5'-GCGTACGCAAAAAAATCCTCCA-3';                                                                        17                                                     5'-TTCGCGGCGTTCGGTTC-3';                                                                             18                                         Unmethylated VHL                                                                          5'-ACATACACAAAAAAATCCTCCAAC-3';                                                                      19                                                     5'-TTTGTGGTGTTTGGTTTGGG-3';                                                                          20                                         Methylated E-cadherin                                                                     5'-ACGCGATAACCCTCTAACCTAA-3';                                                                        21                                                     5'-GTCGGTAGGTGAATTTTTAGTTA-3';                                                                       22                                         Unmethylated E-cadherin                                                                   5'-ACAATAACCCTCTAACCTAAAATTA-3'; and                                                                 23                                                     5'-TGTGTTGTTGATTGGTTGTG-3'.                                                                          24                                         __________________________________________________________________________

Exemplary primer pairs included in the invention that hybridize to theabove sequences include:

    ______________________________________                                                               SEQ ID NO:                                             ______________________________________                                        5'-CAGAGGGTGGGGCGGACCGC-3' and                                                                         26                                                   5'-CGGGCCGCGGCCGTGG-3';  27                                                   5'-TTATTAGAGGGTGGGGCGGATCGC-3' and                                                                     28                                                   5'-GACCCCGAACCGCGACCGTAA-3';                                                                           29                                                   5'-TTATTAGAGGGTGGGGTGGATTGT-3' and                                                                     30                                                   5'-CAACCCCAAACCACAACCATAA-3';                                                                          31                                                   5'-TTATTAGAGGGTGGGGCGGATCGC-3' and                                                                     32                                                   5-CCACCTAAATCGACCTCCGACCG-3';                                                                          33                                                   5'-TTATTAGAGGGTGGGGTGGATTGT-3' and                                                                     34                                                   5'-CCACCTAAATCAACCTCCAACCA-3';                                                                         35                                                   5'-CGCACCCTGCGGCCAGA-3' and                                                                            36                                                   5'-AGTGGCCGAGCGGCCGG-3'; 37                                                   5'-GCGTTCGTATTTTGCGGTT-3' and                                                                          38                                                   5'-CGTACAATAACCGAACGACCGA-3';                                                                          39                                                   5'-TGTGATGTGTTTGTATTTTGTGGTT-3' and                                                                    40                                                   5'-CCATACAATAACCAAACAACCAA-3';                                                                         41                                                   5'-TGGAGGATTTTTTTGCGTACGC-3' and                                                                       42                                                   5'-GAACCGAACGCCGCGAA-3'; 43                                                   5'-GTTGGAGGATTTTTTTGTGTATGT-3' and                                                                     44                                                   5'-CCCAAACCAAACACCACAAA-3';                                                                            45                                                   5'-TTAGGTTAGAGGGTTATCGCGT-3' and                                                                       46                                                   5'-TAACTAAAAATTCACCTACCGAC-3'; and                                                                     47                                                   5'-TAATTTTAGGTTAGAGGGTTATTGT-3' and                                                                    48                                                   5'-CACAACCAATCAACAACACA-3'.                                                                            49                                                   ______________________________________                                         *Also included are modifications of the above sequences, including SEQ ID     NO:26 having the sequence TCAC at the 5' end; SEQ ID NO:27 having the         sequence CC added at the 5' end; SEQ ID NO:28 having the sequence             5TTATTAGAGGGTGGGGCGGATCGC-3'; SEQ ID NO:29 having the sequence                5GACCCCGAACCGCGACCGTAA-3'; SEQ ID NO:30 having the sequence TGG added at      the 5' end; and SEQ ID NO:31 having the sequence TACC added at the 5' end     All of these modified primers  anneal at 65° C.                   

Typically, the CpG-containing nucleic acid is in the region of thepromoter of a structural gene. For example, the promoter region of tumorsuppressor genes have been identified as containing methylated CpGisland. The promoter region of tumor suppressor genes, including p16,p15, VHL and E-cadherin, are typically the sequence amplified by PCR inthe method of the invention.

Detection and identification of methylated CpG-containing nucleic acidin the specimen may be indicative of a cell proliferative disorder orneoplasia. Such disorders include but are not limited to low gradeastrocytoma, anaplastic astrocytoma, glioblastoma, medulloblastoma,colon cancer, lung cancer, renal cancer, leukemia, breast cancer,prostate cancer, endometrial cancer and neuroblastoma. Identification ofmethylated CpG status is also useful for detection and diagnosis ofgenomic imprinting, fragile X syndrome and X-chromosome inactivation.

The method of the invention now provides the basis for a kit useful forthe detection of a methylated CpG-containing nucleic acid. The kitincludes a carrier means being compartmentalized to receive in closeconfinement therein one or more containers. For example, a firstcontainer contains a reagent which modifies unmethylated cytosine, suchas sodium bisulfite. A second container contains primers foramplification of the CpG-containing nucleic acid, for example, primerslisted above for p16, p15, VHL or E-cadherin.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly and are not intended to limit the scope of the invention.

EXAMPLE 1

DNA and Cell Lines. Genomic DNA was obtained from cell lines, primarytumors and normal tissue as described (Merlo, et al., Nature Medicine,1:686, 1995; Herman, et al., Cancer Research, 56:722, 1996; Graff, etal., Cancer Research, 55:5195, 1995). The renal carcinoma cell line waskindly provided by Dr. Michael Lehrman of the National Cancer Institute,Bethesda, MD.

Bisulfite Modification. 1 μg of DNA in a volume of 50 μl was denaturedby NaOH (final 0.2M) for 10 minutes at 37° C. For samples with nanogramquantities of human DNA, 1 μg of salmon sperm DNA (Sigma) was added ascarrier prior to modification. 30 μL of 10 mM hydroquinone (Sigma) and520 μL of 3M sodium bisulfite (Sigma) pH5, both freshly prepared, wereadded, mixed, and samples were incubated under mineral oil at 50° C. for16 hours. Modified DNA was purified using the Wizard™ DNA purificationresin according to the manufacturer (Promega), and eluted into 50 μL ofwater. Modification was completed by NaOH (final 0.3M) treatment for 5minutes at room temperature, followed by ethanol precipitation.

Genomic Sequencing. Genomic sequencing of bisulfite modified DNA wasaccomplished using the solid-phase DNA sequencing approach (Myohanen, etal., DNA Seq., 5:1, 1994). 100 ng of bisulfite modified DNA wasamplified with p16 gene specific primer 5'-TTTTTAGAGGATTTGAGGGATAGG-3'(sense) (SEQ ID NO:49) and 5'-CTACCTAATTCCAATTCCCCTACA-3' (anti-sense)(SEQ ID NO:50). PCR conditions were as follows: 96° C. for 3 minutes,80° C. for 3 minutes, 1 U of Taq polymerase (BRL) was added, followed by35 cycles of 96° C. for 20 seconds, 56° C. for 20 seconds, 72° C. for 90seconds, followed by 5 minutes at 72° C. The PCR mixture contained 1Xbuffer (BRL) with 1.5 mM MgCl₂, 20 pmols of each primer and 0.2 mMdNTPs. To obtain products for sequencing, a second round of PCR wasperformed with 5 pmols of nested primers. In this reaction, the senseprimer, 5'-GTTTTCCCAGTCACGACAGTATTAGGAGG AAG AAAGAGGAG-3' (SEQ IDNO:51), contains M13-40 sequence (underlined) introduced as a site toinitiate sequencing, and the anti-sense primer5'-TCCAATTCCCCTACAAACTTC-3" (SEQ ID NO:52) is biotinylated to facilitatepurification of the product prior to sequencing. PCR was performed asabove, for 32 cycles with 2.5 mM MgCl₂. All primers for genomicsequencing were designed to avoid any CpGs in the sequence. BiotinylatedPCR products were purified using streptavidin coated magnetic beads(Dynal AB, Norway), and sequencing reactions performed with Sequenase™and M13-40 sequencing primer under conditions specified by themanufacturer (USB).

PCR Amplification. Primer pairs described in Table 1 were purchased fromLife Technologies. The PCR mixture contained 1X PCR buffer (16.6 mMammonium sulfate, 67 mM TRIS pH 8.8, 6.7 mM MgCl₂, and 10 mMβ-mercaptoethanol), dNTPs (each at 1.25 mM), primers (300 ng/reactioneach), and bisulfite-modified DNA (˜50 ng) or unmodified DNA (50-100 ng)in a final volume of 50 μL. PCR specific for unmodified DNA alsoincluded 5% dimethylsulfoxide. Reactions were hot started at 95° C. for5 minutes prior to the addition of 1.25 units of Taq polymerase (BRL).Amplification was carried out on a Hybaid OmniGene temperature cyclerfor 35 cycles (30 seconds at 95° C., 30 seconds at the annealingtemperature listed in Table 1, and 30 seconds at 72° C.), followed by afinal 4 minute extension at 72° C. Controls without DNA were performedfor each set of PCR reactions. 10 μL of each PCR reaction was directlyloaded onto non-denaturing 6-8% polyacrylamide gels, stained withethidium bromide, and directly visualized under UV illumination.

Restriction Analysis. 10 μL of the 50 μL PCR reaction was digested with10 units of BstUI (New England Biolabs) for 4 hours according toconditions specified by the manufacturer. Restriction digests wereethanol precipitated prior to gel analysis.

EXAMPLE 2

An initial study was required to validate the strategy for MSP forproviding assessment of the methylation status of CpG islands. The p16tumor suppressor (Merlo, et al., supra; Herman, et al., Cancer Research,55:4525, 1995; Gonzalez-Zulueta, et al., Cancer Res., 55:4531, 1995,27)which has been documented to have hypermethylation of a 5' CpG island isassociated with complete loss of gene expression in many cancer types,was used as an exemplary gene to determine whether the density ofmethylation, in key regions to be tested, was great enough to facilitatethe primer design disclosed herein. Other than for CpG sites located inrecognition sequences for methylation-sensitive enzymes, the density ofmethylation and its correlation to transcriptional silencing had not yetbeen established. The genomic sequencing technique was thereforeemployed to explore this relationship.

FIG. 1 shows genomic sequencing of p16. The sequence shown has the most5' region at the bottom of the gel, beginning at +175 in relation to amajor transcriptional start site (Hara, et al., Mol. Cell Biol., 16:859,1996). All cytosines in the unmethylated cell line H249 have beenconverted to thymidine, while all C's in CpG dinucleotides in themethylated cell H157 remains as C, indicating methylation. ! enclosed aBstUI site which is at -59 in relation to the transnational start sitein Genbank sequence U12818 (Hussussian, et al., Nat. Genet., 8:15,1994), but which is incorrectly identified as CGCA in sequence X94154(Hara, et al., supra). This CGCG site represents the 3' location of thesense primer used for p16 MSP.

As has been found for other CpG islands examined in this manner(Myohanen, et al., supra; Park, et al., Mol. Cell Biol., 14:7975, 1994;Reeben, et al., Gene, 157:325, 1995), the CpG island of p16 wascompletely unmethylated in those cell lines and normal tissuespreviously found to be unmethylated by Southern analysis (FIG. 1)(Merlo,et al., supra; Herman, et al, supra). However, it was extensivelymethylated in cancer cell lines shown to be methylated by Southernanalysis (FIG. 1). In fact, all cytosines within CpG dinucloetides inthis region were completely methylated in the cancers lacking p16transcription. This marked difference in sequence following bisulfitetreatment suggested that the method of the invention for specificamplification of either methylated or unmethylated alleles was usefullfor identification of methylation patterns in a DNA sample.

Primers were designed to discriminate between methylated andunmethylated alleles following bisulfite treatment, and to discriminatebetween DNA modified by bisulfite and that which had not been modified.To accomplish this, primer sequences were chosen for regions containingfrequent cytosines (to distinguish unmodified from modified DNA), andCpG pairs near the 3' end of the primers (to provide maximaldiscrimination in the PCR reaction between methylated and unmethylatedDNA). Since the two strands of DNA are no longer complementary afterbisulfite treatment, primers can be designed for either modified strand.For convenience, primers were designed for the sense strand. Thefragment of DNA to be amplified was intentionally small, to allow theassessment of methylation patterns in a limited region and to facilitatethe application of this technique to samples, such as paraffin blocks,where amplification of larger fragments is not possible. In Table 1,primer sequences are shown for all genes tested, emphasizing thedifferences in sequence between the three types of DNA which areexploited for the specificity of MSP. The multiple mismatches in theseprimers which are specific for these different types of DNA suggest thateach primer set should provide amplification only from the intendedtemplate.

The primers designed for p16 were tested with DNA from cancer cell linesand normal tissues for which the methylation status had previously beendefined by Southern analysis (Merlo, et al., supra; Herman, et al.,supra).

FIG. 2, panels A-D, show polyacrylamide gels with the MethylationSpecific PCR products of p16. Primer sets used for amplification aredesignated as unmethylated (U), methylated (M), or unmodified/wild-type(W).* designates the molecular weight marker pBR322-MspI digest. Panel Ashows amplification of bisulfite-treated DNA from cancer cell lines andnormal lymphocytes, and untreated DNA (from cell line H249). Panel Bshows mixing of various amount of H157 DNA with 1 μg of H249 DNA priorto bisulfite treatment to assess the detection sensitivity of MSP formethylated alleles. Modified DNA from a primary lung cancer sample andnormal lung are also shown. Panel C shows amplification with the p16-U2(U) primers, and p16-M2 (M) described in Table 1. Panel D shows theamplified p16 products of panel C restricted with BstUI(+) or notrestricted (-). In all cases, the primer set used confirmed themethylation status determined by Southern analysis. For example, lungcancer cell lines U1752 and H157, as well other cell lines methylated atp16, amplified only with the methylated primers (FIG. 2, panel A). DNAfrom normal tissues (lymphocytes, lung, kidney, breast, and colon) andthe unmethylated lung cancer cell lines H209 and H249, amplified onlywith unmethylated primers (examples in FIG. 2, panel A). PCR with theseprimers could be performed with or without 5% DMSO. DNA not treated withbisulfite (unmodified) failed to amplify with either set of methylatedor unmethylated specific primers, but readily amplified with primersspecific for the sequence prior to modification (FIG. 2, panel A). DNAfrom the cell line H157 after bisulfite treatment also produced a weakeramplification with unmodified primers, suggesting an incompletebisulfite reaction. However, this unmodified DNA, unlike partiallyrestricted DNA in previous PCR assays relying on methylation sensitiverestriction enzymes, is not recognized by the primers specific formethylated DNA. It therefore does not provide a false positive result orinterfere with the ability to distinguish methylated from unmethylatedalleles.

The sensitivity of MSP for detection of methylated p16 alleles wasassessed. DNA from methylated cell lines was mixed with unmethylated DNAprior to bisulfite treatment. 0.1% of riethylated DNA (approximately 50pg) was consistently detected in an otherwise unmethylated sample (FIG.2, panel B). The sensitivity limit for the amount of input DNA wasdetermined to be as little as 1 ng of human DNA, mixed with salmon spermDNA as a carrier detectable by MSP.

Fresh human tumor samples often contain normal and tumor tissue, makingthe detection of changes specific for the tumor difficult. However, thesensitivity of MSP suggests it would be useful for primary tumors aswell, allowing for detection of aberrantly methylated alleles even ifthey contribute relatively little to the overall DNA in a sample. Ineach case, while normal tissues were completely unmethylated, tumorsdetermined to be methylated at p16 by Southern analysis also containedmethylated DNA detected by MSP, in addition to some unmethylated alleles(examples in FIG. 2, panel B). DNA from paraffin-embedded tumors wasalso used, and allowed the detection of methylated and unmethylatedalleles in these samples (FIG. 2, panel B). To confirm that theseresults were not unique to this primer set, a second downstream primerfor p16 was used which would amplify a slightly larger fragment (Table1). This second set of primers reproduced the results described above(FIG. 2, panel C), confirming the methylation status defined by Southernblot analysis.

To further verify the specificity of the primers for the methylatedalleles and to check specific cytosines for methylation within theregion amplified, the differences in sequence betweenmethylated/modified DNA and unmethylated/modified DNA were utilized.Specifically, the BstUI recognition site, CGCG, will remain CGCG if bothC's are methylated after bisulfite treatment and amplification, but willbecome TGTG if unmethylated. Digestion of the amplified products withBstUI distinguishes these two products. Restriction of p16 amplifiedproducts illustrates this. Only unmodified products andmethylated/modified products, both of which retain the CGCG site, werecleaved by BstUI, while products amplified with unmethylated/modifiedprimers failed to be cleaved (FIG. 2, panel D).

The primer sets discussed above were designed to discriminate heavilymethylated CpG islands from unmethylated alleles. To do this, both theupper (sense) and lower (antisense) primers contained CpG sites whichcould produce methylation-dependent sequence differences after bisulfitetreatment. MSP might be employed to examine more regional aspects of CpGisland methylation. To examine this, methylation-dependent differencesin the sequence of just one primer was tested to determine whether itwould still allow discrimination between unmethylated and methylatedpp16 alleles. The antisense primer used for genomic sequencing,5'-CTACCTAATTCCAATTCCCCTACA-3' (SEQ ID NO:53), was also used as theantisense primer, since the region recognized by the primer contains noCpG sites, and was paired with either a methylated or unmethylated senseprimer (Table 1). Amplification of the 313 bp PCR product only occurredwith the unmethylated sense primer in H209 and H249 (unmethylated bySouthern) and the methylated sense primer in H157 and U1752 (methylatedby Southern), indicating that methylation of CpG sites within a definedregion can be recognized by specific primers and distinguish betweenmethylated and unmethylated alleles (FIG. 2, panel E). Panel E showsresults of testing for regional methylation of CpG islands with MSP,using sense primers p16-U2 (U) and p16-M2 (M), which are methylationspecific, and an antisense primer which is not methylation specific.

EXAMPLE 3

The above experiments with p16 were extended to include 3 other genestranscriptionally silenced in human cancers by aberrant hypermethylationof 5' CpG islands.

FIG. 3, panels A-E, show polyacrylamide gels of MSP products fromanalysis of several genes. Primer sets used for amplification are notdesignated as unmethylated (U), methylated (M), or unmodified/wild-type(W). * designates the molecular weight marker pBR322-MspI digest and **designates the 123 bp molecular weight marker. All DNA samples werebisulfite treated except those designated untreated. Panel A shows theresults from MSP for p15. Panel B shows the p15 products restricted withBstUI (+) or not restricted (-). Panel C shows the products of MSP forVHL. Panel D shows the VHL products restricted with BstUI(+) or notrestricted (-). Panel E shows the products of MSP for E-cadherin.

The cyclin-dependent kinase inhibitor p15 is aberrantly methylated inmany leukemic cell lines and primary leukemias (Herman, et al., supra).For p15, MSP again verified the methylation status determined bySouthern analysis. Thus, normal lymphocytes and cancer cell lines SW48and U1752, all unmethylated by Southern analysis (Herman, et al, supra),only amplified with the unmethylated set of primers, while the lungcancer cell line H1618 and leukemia cell line KG1A amplified only withthe methylated set of primers (FIG. 3, panel A), consistent withprevious Southern analysis results (Herman, et al., supra). The cellline Raji produced a strong PCR product with methylated primers and aweaker band with unmethylated primers. This was the same result formethylation obtained previously by Southern analysis (Herman, et al.,supra). Non-cultured leukemia samples, like the primary tumors studiedfor p16, had amplification with the methylated primer set as well as theunmethylated set. This heterogeneity also matched Southern analysis(Herman, et al., supra). Again, as for p16, differential modification ofBstUI restriction sites in the amplified product of p15 was used toverify the specific amplification by MSP (FIG. 3, panel B). Amplifiedproducts using methylated primer sets from cell lines H1618 and Raji orunmodified primer sets, were completely cleaved by BstUI, whileunmethylated amplified products did not cleave. Primary AML samples,which again only demonstrated cleavage in the methylated product, hadless complete cleavage. This suggests a heterogeneity in methylation,arising because in some alleles, many CpG sites within the primersequences area are methylated enough to allow the methylation specificprimers to amplify this region, while other CpG sites are not completelymethylated.

Aberrant CpG island promoter region methylation is associated withinactivation of the VHL tumor suppressor gene in approximately 20% ofclear renal carcinomas (Herman, et al., Proc. Natl. Acad. Sci. USA,91:9700, 1994). This event, like mutations for VHL (Gnarra, et al.,Nature Genetics, 7:85, 1994), is restricted to clear renal cancers(Herman, et al, supra). Primers designed for the VHL sequence were usedto study DNA from the renal cell cancer line RFX393 which is methylatedat VHL by Southern analysis, and the lung cancer cell line U1752 whichis unmethylated at this locus (Herman, et al., supra). In each case, themethylation status of VHL determined by MSP confirmed that found bySouthern analysis (FIG. 3, panel C), and BstUI restriction site analysisvalidated the PCR product specificity (FIG. 3, panel D).

The expression of the invasion/metastasis suppressor gene, E-cadherin,is often silenced by aberrant methylation of the 5' promoter in breast,prostate, and many other carcinomas (Graff, et al., supra; Yoshira, etal., Proc. Natl. Acad. Sci. USA, 92:7416, 1995). Primers were designedfor the E-cadherin promoter region to test the use of MSP for this gene.In each case, MSP analysis paralleled Southern blot analysis for themethylation status of the gene (Graff, et al., supra). The breast cancercell lines MDA-MB-231, HS578t, and the prostate cancer cell lines DuProand TSUPrI, all heavily methylated by Southern, displayed prominentmethylation. MCF7, T47D, PC-3, and LNCaP, all unmethylated by Southern,showed no evidence for methylation in the sensitive MSP assay (FIG. 3,panel E). MSP analysis revealed the presence of unmethylated alleles inHs578t, TSUPrI and DuPro consistent with a low percentage ofunmethylated alleles in these cell lines previously detected by Southernanalysis (Graff, el al., supra). BstUI restriction analysis againconfirmed the specificity of the PCR amplification.

                                      TABLE 1                                     __________________________________________________________________________    PCR primers used for Methylation Specific PCR                                 Primer                                 Size                                                                             Anneal                                                                            Genomic                         Set Sense primer* (5'-3')                                                                           Antisense primer* (5'-3')                                                                      (bp)                                                                             temp.                                                                             Position†                __________________________________________________________________________    p16-W†                                                                     CAGAGGGTGGGGCGACCGC                                                                             CGGGCCGCGGCCGTGG 140                                                                              65° C.                                                                     +171                            p16-M                                                                             TTATTAGAGGGTGGGGCGGATCGC                                                                        GACCCCGAACCGCGACCGTAA                                                                          150                                                                              65° C.                                                                     +167                            p16-U                                                                             TTATTAGAGGGTGGGGTGGATTGT                                                                        CAACCCCAAACCACAACCATAA                                                                         151                                                                              60° C.                                                                     +167                            p16-M2                                                                            TTATTAGAGGGTGGGGCGGATCGC                                                                        CCACCTAAATCGACCTCCGACCG                                                                        234                                                                              65° C.                                                                     +167                            p16-U2                                                                            TTATTAGAGGGTGGGGTGGATTGT                                                                        CCACCTAAATCAACCTCCAACCA                                                                        234                                                                              60° C.                                                                     +167                            p15-W                                                                             CGCACCCTGCGGCCAGA AGTGGCCGAGCGGCCGG                                                                              137                                                                              65° C.                                                                     +46                             p15-M                                                                             GCGTTCGTATTTTGCGGTT                                                                             CGTACAATAACCGAACGACCGA                                                                         148                                                                              60° C.                                                                     +40                             p15-U                                                                             TGTGATGTGTTTGTATTTTGTGGTT                                                                       CCATACAATAACCAAACAACCAA                                                                        154                                                                              60° C.                                                                     +34                             VHL-M                                                                             TGGAGGATTTTTTTGCGTACGC                                                                          GAACCGAACGCCGCGAA                                                                              158                                                                              60° C.                                                                     -116                            VHL-U                                                                             GTTGGAGGATTTTTTTGTGTATGT                                                                        CCCAAACCAAACACCACAAA                                                                           165                                                                              60° C.                                                                     -118                            Ecad-M                                                                            TTAGGTTAGAGGGTTATCGCGT                                                                          TAACTAAAAATTCACCTACCGAC                                                                        116                                                                              57° C.                                                                     -205                            Ecad-U                                                                            TAATTTTAGGTTAGAGGGTTATTGT                                                                       CACAACCAATCAACAACACA                                                                            97                                                                              53° C.                                                                     -210                            __________________________________________________________________________     *Sequence differences between modified primers and unmodified DNA are         boldface, and differences between methylated/modified acid                    unmethylated/modified are underlined.                                         †Primers were placed near the transcriptional strat site. Genomic      position is the location of the 5' nucleotide of the sense primer in          relation to the major transcriptional start site defined in the following     refercnces and Genbank accession numbers: p16(most 3' site) X94154 (E.        Hara, et al., Mol. Cell Biol., 16:859, 1996), p15 S75756 (J. Jen, et al.,     Cancer Res., 54: 6353 1994), VHL U19763 (I. Kuzmin, et al., Oncogene,         10:2185 1995), and Ecadherin 34545 (M. J. Bussemakers, et al., Biochem.       Biophys. Res. Commun., 203: 1284 1994).                                       †W represents unmodified, or wildtype primers, M represents            methylatedspecific primers, and U represents unmethylatedspecific primers     (SEQ ID NO:26-48)                                                        

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 52                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCGGTCCGCCCCACCCTCTG20                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CCACGGCCGCGGCCCG16                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCGATCCGCCCCACCCTCTAATAA24                                                    (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TTACGGTCGCGGTTCGGGGTC21                                                       (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ACAATCCACCCCACCCTCTAATAA24                                                    (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TTATGGTTGTGGTTTGGGGTTG22                                                      (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) 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        (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ACAATCCACCCCACCCTCTAATAA24                                                    (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TGGTTGGAGGTTGATTTAGGTGG23                                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TCTGGCCGCAGGGTGCG17                         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DESCRIPTION: SEQ ID NO:14:                                      TCGGTCGTTCGGTTATTGTACG22                                                      (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AACCACAAAATACAAACACATCACA25                                                   (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      TTGGTTGTTTGGTTATTGTATGG23                                                     (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GCGTACGCAAAAAAATCCTCCA22                                                      (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TTCGCGGCGTTCGGTTC17                                                           (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      ACATACACAAAAAAATCCTCCAAC24                                                    (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TTTGTGGTGTTTGGTTTGGG20                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ACGCGATAACCCTCTAACCTAA22                                                      (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GTCGGTAGGTGAATTTTTAGTTA23                                                     (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      ACAATAACCCTCTAACCTAAAATTA25                                                   (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      TGTGTTGTTGATTGGTTGTG20                                                        (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      CAGAGGGTGGGGCGGACCGC20                                                        (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      CGGGCCGCGGCCGTGG16                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      TTATTAGAGGGTGGGGCGGATCGC24                                                    (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GACCCCGAACCGCGACCGTAA21                                                       (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TTATTAGAGGGTGGGGTGGATTGT24                                                    (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CAACCCCAAACCACAACCATAA22                                                      (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      TTATTAGAGGGTGGGGTGGATTGT24                                                    (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      CCACCTAAATCGACCTCCGACCG23                                                     (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      TTATTAGAGGGTGGGGTGGATTGT24                                                    (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CCACCTAAATCAACCTCCAACCA23                                                     (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      CGCACCCTGCGGCCAGA17                                                           (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      AGTGGCCGAGCGGCCGG17                                                           (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GCGTTCGTATTTTGCGGTT19                                                         (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      CGTACAATAACCGAACGACCGA22                                                      (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      TGTGATGTGTTTGTATTTTGTGGTT25                                                   (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      CCATACAATAACCAAACAACCAA23                                                     (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      TGGAGGATTTTTTTGCGTACGC22                                                      (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      GAACCGAACGCCGCGAA17                                                           (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      GTTGGAGGATTTTTTTGTGTATGT24                                                    (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      CCCAAACCAAACACCACAAA20                                                        (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      TTAGGTTAGAGGGTTATCGCGT22                                                      (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      TAACTAAAAATTCACCTACCGAC23                                                     (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      TAATTTTAGGTTAGAGGGTTATTGT25                                                   (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      CACAACCAATCAACAACACA20                                                        (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      TTTTTAGAGGATTTGAGGGATAGG24                                                    (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      CTACCTAATTCCAATTCCCCTACA24                                                    (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      GTTTTCCCAGTCACGACAGTATTAGGAGGAAGAAAGAGGAG41                                   (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      TCCAATTCCCCTACAAACTTC21                                                       __________________________________________________________________________

What is claimed is:
 1. A method for detecting a methylatedCpG-containing nucleic acid comprising:contacting a nucleicacid-containing specimen with an agent that modifies unmethylatedcytosine, amplifying the CpG-containing nucleic acid in the specimen bymeans of CpG-specific oligonucleotide primers, wherein theoligonucleotide primers distinguish between modified methylated andnonmethylated nucleic acid, and detecting the methylated nucleic acidbased on the presence or absence of amplification products produced insaid amplifying step.
 2. The method of claim 1, wherein the amplifyingstep is the polymerase chain reaction (PCR).
 3. The method of claim 1,wherein the modifying agent is bisulfite.
 4. The method of claim 1,wherein cytosine is modified to uracil.
 5. The method of claim 1,wherein the CpG-containing nucleic acid is in a promoter region.
 6. Themethod of claim 5, wherein the promoter is a tumor suppressor genepromoter.
 7. The method of claim 6, wherein the tumor suppressor gene isselected from the group consisting of p16, p15, E-cadherin, and VHL. 8.The method of claim 1, wherein the specimen is from a tissue selectedfrom the group consisting of brain, colon, urogenital, lung, renal,hematopoietic, breast, thymus, testis, ovarian, and uterine.
 9. Themethod of claim 1, further comprising contacting the nucleic acid with amethylation sensitive restriction endonuclease.
 10. The method of claim9, wherein the restriction endonuclease is selected from the groupconsisting ofMspI, HpaII, BssHII, BstUI and NotI.
 11. The method ofclaim 1, wherein the presence of methylated CpG-containing nucleic acidin the specimen is indicative of a cell proliferative disorder.
 12. Themethod of claim 11, wherein the disorder is selected from the groupconsisting of low grade astrocytoma, anaplastic astrocytoma,glioblastoma, medulloblastoma, colon cancer, lung cancer, renal cancer,leukemia, breast cancer, prostate cancer, endometrial cancer andneuroblastoma.
 13. The method of claim 1, wherein the primer hybridizeswith a target polynucleotide sequence having the sequence selected fromthe group consisting of SEQ ID NO:1-23 and SEQ ID NO:24.
 14. The methodof claim 1, wherein the primers are selected from the group consistingof SEQ ID NO:25-47 and SEQ ID NO:48.
 15. A kit useful for the detectionof a methylated CpG-containing nucleic acid comprising carrier meansbeing compartmentalized to receive in close confinement therein one ormore containers comprising a first container containing a reagent whichmodifies unmethylated cytosine and a second container containing primersfor amplification of the CpG-containing nucleic acid, wherein theprimers distinguish between modified methylated and nonmethylatednucleic acid.
 16. The kit of claim 15, wherein the modifying reagent isbisulfite.
 17. The kit of claim 15, wherein said reagent modifiescytosine to uracil.
 18. The kit of claim 15, wherein the primerhybridizes with a target polynucleotide sequence having the sequenceselected from the group consisting of SEQ ID NO:1-23 and SEQ ID NO:24.19. The kit of claim 15, wherein the primers are selected from the groupconsisting of SEQ ID NO:25-47 and
 48. 20. Isolated oligonucleotideprimer(s) for detection of a methylated CpG-containing nucleic acidwherein the primer hybridizes with a target polynucleotide sequencehaving the sequence selected from the group consisting of SEQ ID NO:1-23and SEQ ID NO:24.
 21. The primers of claim 20, wherein the primer pairsare selected from the group consisting of SEQ ID NO:25-47 and
 48. 22. Akit for the detection of methylated CpG-containing nucleic acid from asample comprising:a) a reagent that modifies unmethylated cytosinenucleotides; b) a wild-type unmodified control nucleic acid; c) primersfor the amplification of unmethylated CpG-containing nucleic acid; d)primers for the amplification of methylated CpG-containing nucleic acid;and e) primers for the amplification of control unmodified nucleic acid,wherein the primers for the amplification of unmethylated CpG-containingnucleic acid and methylated CpG-containing nucleic acid distinguishbetween modified methylated and nonmethylated nucleic acid.
 23. The kitof claim 22, further comprising nucleic acid amplification buffer. 24.The kit of claim 22, wherein the reagent that modifies unmethylatedcytosine is bisulfite.
 25. The kit of claim 22, wherein primershybridize with a target polynucleotide sequence having the sequenceselected from the group consisting of SEQ ID NO:1-23 and SEQ ID NO:24.26. The kit of claim 22, wherein the primers are selected from the groupconsisting of SEQ ID NO:25-47 and SEQ ID NO:48.
 27. A method fordetecting a methylated CpG-containing nucleic acid comprising:contactinga nucleic acid-containing specimen with bisulfite to modify unmethylatedcytosine, amplifying the CpG-containing nucleic acid in the specimen bymeans of CpG-specific oligonucleotide primers, wherein theoligonucleotide primers distinguish between modified methylated andnonmethylated nucleic acid and detecting the methylated nucleic acidbased on the presence or absence of amplification products produced insaid amplifying step.